The invention relates to the production of follicle stimulating hormone (FSH) with altered glycosylation patterns and activities. In particular, it concerns production of recombinant FSH under conditions which regulate the glycosylation pattern of the protein.
Human FSH is used therapeutically to regulate various aspects of metabolism pertinent to reproduction in the human female. For example, FSH partially purified from urine is used clinically to stimulate follicular maturation in anovulatory women with anovulatory syndrome or luteal phase deficiency. It is also used in combination with luteinizing hormone (LH) to stimulate the development of ovarian follicles for in vitro fertilization. The role of FSH in the reproductive cycle is sufficiently well-known to permit this sort of therapeutic use, but difficulties have been encountered due, in part, to the heterogeneity of the preparation from native sources. This heterogeneity is due to variations in glycosylation pattern.
FSH is one member of a family of heterodimeric human glycoprotein hormones which have a common alpha subunit, but differ in their hormone-specific beta subunits. The family includes, besides FSH, luteinizing hormone (LH), thyrotropin or thyroid stimulating hormone (TSH), and human chorionic gonadotropin (CG). In all cases, the alpha subunit is a 92 amino acid glycoprotein with two canonical glycosylation sites at the asparagines located at positions 52 and 78. The beta subunits are also glycoproteins; in addition to the N-linked glycosylation exhibited by the beta chains of all four hormones, human CG contains four mucin-like O-linked oligosaccharides attached to a carboxy-terminal extension unique to this hormone. The relevance of the O-linked glycosylation is not, apparently, related to the secretion and assembly of the hormone (Matzuk, M. M. et al. Proc Natl Acad Sci USA (1987) 84:6354-6358).
Genomic and cDNA clones have been prepared corresponding to the human alpha chain (Boothby, M. et al. J Biol Chem (1981) 256:5121-5127; Fiddes, J. C. et al. J Mol App Genet (1981) 1:3-18). The cDNA and genomic sequences of the beta subunits of the remaining three members of the family have also been prepared: for CG, as disclosed by Fiddes, J. C. et al. Nature (1980) 286:684-687 and by Policastro, P. et al. J Biol Chem (1983) 258:11492-11499; for luteinizing hormone by Boorstein, W. R. et al. Nature (1982) 300:419-422; and for TSH by Hayashizaki, Y. et al. FEBS Lett (1985) 188:394-400 and by Whitfield, G. K. et al. in xe2x80x9cFrontiers in Thyroidologyxe2x80x9d, (1986) Medeiros-Nato, G. et al. (eds) pages 173-176, Plenum Press, N. Y. These DNA segments have been expressed recombinantly, and biologically active material has been produced.
Although genomic clones and isolates for human FSH-beta hve been prepared (Watkins, P. C. et al. DNA (1987) 6:205-212; Jameson, J. L. et al., Mol Endocrinol (1988) 2:806-815; Jameson, J. L. et al. J Clin Endocrinol Metab (1986) 64:319-327; Glaser, T. et al. Nature (1986) 321:882-887), human FSH beta has not been engineered to permit recombinant production of the hormone. (The bovine beta FSH gene has also been obtained as disclosed in Maurer, R. A. et al. DNA (1986) 5:363-369; Kim, K. E. et al. DNA (1988) 7:227-333.) As disclosed in the invention herein, recombinant production of this FSH hormone permits regulation of the glycosylation pattern and thereby greater predictability in the formulation of therapeutically useful material.
While it is now understood that the glycosylation pattern of a particular protein may have considerable relevance to its biological activity, the importance of this pattern has largely been overlooked in characterization of glycoproteins. Emphasis has been placed on the amino acid sequence as if this were the sole component of the glycoprotein. The reasons for this myopia are largely historic, but this almost exclusive focus on the peptide aspect is clearly in error. For example, it is well known in the case of human CG that desialylation causes the hormone to be cleared rapidly via the liver (Morell, A. G. et al. J Biol Chem (1971) 246:1461-1467). It is also known that removal of carbohydrate internal to the sialic acid residues or complete deglycosylation converts human CG into an antagonist which binds more tightly to receptor but shows decreased biological activity in vitro (Channing, C.P. et al. Endocrinol (1978) 103:341-348; Kalyan, N. J. et al. J Biol Chem (1983) 258:67-74; Keutmann, H. T. et al. Biochemistry (1983) 3067-3072; Moyle, W. R. et al. J Biol Chem (1975) 250:9163-9169). Other glycoproteins, such as, for example, tissue plasminogen activator, are also known to be altered in their degree of activity when the glycosylation pattern is changed. Therefore, it appears that in order to regulate the therapeutic function of the glycoprotein hormones, it may be necessary to control both the level and nature of glycosylation.
Disclosure of the Invention
The invention provides recombinantly produced human FSH which offers the opportunity for control of gly-cosylation pattern both on the alpha and beta portions of the heterodimer. Such glycosylation control can be obtained through either the prudent selection of the recombinant eucaryotic host, including mutant eucaryotic hosts, or through alteration of glycosylation sites through, for example, site directed mutagenesis at the appropriate amino acid residues. In any event, the recombinant production of this hormone obviates the complex mixture of glycosylation patterns obtained when the hormone is isolated from native sources.
In one aspect, the invention is directed to expression systems capable, when transformed into a suitable host, of expressing the gene encoding the FSH beta subunit. In additional aspects, the invention is directed to recombinant hosts which have been transformed or transfected with this expression system, either singly, or in combination with an expression system capable of producing the alpha subunit. In other aspects, the invention is directed to the FSH beta monomers and FSH heterodimers of defined glycosylation pattern produced by the recombinant host cells.
In another aspect, the invention is directed to specific mutants of FSH or other hormones of this family with altered glycosylation patterns at the two glycosylation sites in the alpha subunit, or to alpha subunit variants containing alterations at the carboxy terminus which affect activity and to glycosylation or other variants of the FSH beta subunit. Thus, in another aspect, the invention is directed to expression systems for the alpha subunit which lack glycosylation sites at the asparagine at position 52 or position 78 or both, for the FSH beta subunit and its variants, and to recombinant host cells transfected with these expression systems. The cells may be transfected with a subunit expression system singly or in combination with an expression system for a suitable alpha or beta subunit. The invention is directed also to the mutant glycoproteins with altered glycosylation or activity patterns produced by these cells.
In other aspects, the invention is directed to pharmaceutical compositions containing the variants set forth above, and to methods to regulate reproductive metabolism in subjects by administration of these glycoproteins or their pharmaceutical compositions.